Interaction of Transducin with Uncoordinated 119 Protein (UNC119)

Abstract

The key visual G protein, transducin undergoes bi-directional translocations between the outer segment (OS) and inner compartments of rod photoreceptors in a light-dependent manner thereby contributing to adaptation and neuroprotection of rods. A mammalian uncoordinated 119 protein (UNC119), also known as Retina Gene 4 protein (RG4), has been recently implicated in transducin transport to the OS in the dark through its interaction with the N-acylated GTP-bound transducin-␣ subunit (G␣ t1 ). Here, we demonstrate that the interaction of human UNC119 (HRG4) with transducin is dependent on the N-acylation, but does not require the GTP-bound form of G␣ t1 . The lipid specificity of UNC119 is unique: UNC119 bound the myristoylated N terminus of G␣ t1 with much higher affinity than a prenylated substrate, whereas the homologous prenyl-binding protein PrBP/␦ did not interact with the myristoylated peptide. UNC119 was capable of interacting with G␣ t1 GDP as well as with heterotrimeric transducin (G t ). This interaction of UNC119 with G t led to displacement of G␤ 1 ␥ 1 from the heterotrimer. Furthermore, UNC119 facilitated solubilization of G t from dark-adapted rod OS membranes. Consistent with these observations, UNC119 inhibited rhodopsin-dependent activation of G t , but had no effect on the GTP-hydrolysis by G␣ t1 . A model for the role of UNC119 in the IS3 OS translocation of G t is proposed based on the UNC119 ability to dissociate G t subunits from each other and the membrane. We also found that UNC119 inhibited activation of G o by D2 dopamine receptor in cultured cells. Thus, UNC119 may play conserved inhibitory role in regulation of GPCR-G protein signaling in non-visual tissues.In rod photoreceptors, exposure to bright light causes translocation of the visual G protein, transducin from the photosensitive outer segments (OS) 2 to the inner compartments of the cells (reviewed in Refs. 1-3). The light-dependent translocation of transducin is thought to play an important role in light-adaptation and neuroprotection (4, 5). Significant advances have been made in understanding the mechanism of this phenomenon. The current evidence supports a simple diffusion model, whereby the activation of transducin by photoexcited rhodopsin (R*) causes dissociation of transducin-␣ (G␣ t1 ) and G␤ 1 ␥ 1 subunits allowing them to diffuse into the inner segment (1-9). However, translocated transducin must return to the OS during dark adaptation to restore rod sensitivity. This retrograde translocation occurs on a relatively slow time scale with a halflife of 2.5 h (4). The precise mechanism of transducin return to the OS in the dark is not known. Formation of heterotrimeric G t in the inner segment (IS) appears to be a prerequisite for correct transport of transducin to the OS. Heterotrimeric G t forms in the IS in the absence of R* following hydrolysis of G␣ t1 -bound GTP. GTP and GTP␥S both caused light-dependent transducin redistribution from the OS in permeabilized retinas, but only GTP-translocated G t returned to the OS i...

Photoreceptor Signaling: Supporting Vision across a Wide Range of Light Intensities

“…Indeed, the rate of spontaneous activation (81) is very close to that of transducin return (56). Another study found that UNC119 is able to pull G␣ t from the membrane-bound transducin trimer, suggesting that no such spontaneous activation would be required (80). Once G␣ t is bound to UNC119, solubilization of G␤ 1 ␥ 1 could be subsequently facilitated by the isoprenoidbinding protein PrBP/␦ (82,83) or by another G␤ 1 ␥ 1 -interacting protein, phosducin, shown to reduce G␤ 1 ␥ 1 membrane affinity (84) and assist G␤ 1 ␥ 1 translocation, at least in the lightinduced direction (85).…”

“…A motor-based mechanism (69,70) is unlikely to help as well because molecular motors are not known to extract lipidated proteins from membranes. A solution to this problem was offered (79) by demonstrating that the knock-out of UNC119, a protein capping the acylated N terminus of G␣ t (79,80), significantly impairs transducin return to the outer segment in the dark. In this mechanism, UNC119 acts by keeping G␣ t apart from G␤ 1 ␥ 1 and by maintaining G␣ t in the soluble form to allow its diffusion to the outer segment.…”

The solution structure of the transducin‐α–uncoordinated 119 protein complex suggests occlusion of the Gβ1γ1‐binding sites

“…The C-terminal domain of UNC119 shares significant sequence and structural homology with the prenyl-binding protein PrBP/δ [10, 11, 15]. Interestingly, unlike PrBP/δ, UNC119 selectively binds N-acylated (myristoylated) proteins, including N-acylated Gα t [11, 16, 17]. Furthermore, UNC119 is capable of interacting with heterotrimeric transducin (G t ).…”

“…Furthermore, UNC119 is capable of interacting with heterotrimeric transducin (G t ). UNC119 effectively displaced Gβ 1 γ 1 from G t and facilitated solubilization of Gα t and Gβ 1 γ 1 from membranes [16]. …”

“…The structure revealed the interactions of the acyl moiety and the first 6 residues of Gα t with the hydrophobic pocket of UNC119. The complementary contacts involving 6 Gα t residues in part explain the selectivity of Gα t /UNC119 interaction [11, 16]. Gα t binding to UNC119 is stronger than that of C. elegans ’ Gα ODR-3, and UNC119 does not bind N-myristoylated recoverin [11, 16].…”